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Antimalarial Drug Discovery: Structural InsightsLunev, Sergey

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Chapter 5

Crystal structure of truncated aspartate tran-

scarbamoylase from Plasmodium falciparum

This chapter has been publishedSergey Lunev, Soraya S. Bosch, Fernando A. Batista, Carsten Wrenger and Matthew R. GrovesActa Crystallogr F Struct Biol Commun. 2016;72:523-33.

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Abstract

De novo pyrimidine biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is un-able to take up pyrimidines from the host. Direct (or indirect) inhibition of de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase (Pf-DHODH), the fourth enzyme of the pathway, has already been shown to be lethal for the parasite. In the second step of the plasmodial pyrimidine synthesis pathway, aspartate and carbamoyl phosphate are condensed to N-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcar-bamoylase (PfATC). In this paper, a 2.5-Å crystal structure of PfATC is reported. The space group of PfATC crystals was determined to be mono-clinic P 21, with unit-cell parameters of a = 87.0, b = 103.8, c = 87.1, α = 90.0, β = 117.7, γ = 90.0. The presented PfATC model shares a high degree of homology with the catalytic domain of E. coli ATC. There is as yet no evidence of existence of the regulatory domain in PfATC. Similarly to E. coli, the PfATC was modeled as homo-trimer where each of the three ac-tive sites is formed on the oligomeric interface. Each active site comprises the residues from two adjacent subunits in the trimer with high degree of evolutional conservation. Here, we also describe the activity loss due to mutagenesis of the key active site residues.

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1. Introduction

According to World Health Organization (WHO) 3.3 billion people world-wide are at risk of being infected with malaria. The disease is considered endemic in more than 100 countries. In 2015 the WHO estimated that there were more than 200 million infections and approximately half a million deaths of malaria, where the majority of death cases occurred amongst children under 5 years old in Africa [1].

Malaria is caused by protozoan parasites of genus Plasmodium (P. fal-ciparum, P. vivax, P. knowlesi, P. malariae and P. ovale curtisi). P. fal-ciparum is responsible for the most dangerous and fatal form of the dis-ease. The complex life cycle of the parasite, as well as the spread of current drug-resistant strains, make malaria treatment highly challenging [2]. There is an urgent need of new anti-malarial agents, making the identifi-cation of new drug targets very important [3-7]

The intraerythrocytic stage of P. falciparum is associated with an extraor-dinary resource uptake from the host cell. Active proliferation during this stage requires a supply of purines and pyrimidines for parasite growth to support the production of DNA and parasite replication. Malaria parasites lack the de novo purine synthesis pathway and take up host cell purines for growth [2, 8]. Inhibition of this pathway was shown to be lethal for P. falciparum in vitro [9, 10]. Early biochemical studies on Plasmodi-um parasites P. berghei [11-13], P. knowlesi [14] and P. lophurae [15, 16] demonstrated the inability of Plasmodium species to metabolize pyrimi-dines. The parasites lack a thymidine kinase, the enzyme responsible for salvaging host thymidine, as was confirmed by the completion of the ge-nome sequence [17]. As the parasite lacks the pyrimidine import pathway [18, 19], pyrimidine biosynthesis pathway has been demonstrated to be major target for antimalarial drug research [20-22].

Plasmodial pyrimidine biosynthesis is affected, directly or indirectly, by many of the current antimalarials [23]. For example, the most widely used current antimalarial drug atovaquone [24] is known to inhibit cy-tochrome b in complex III of the respiratory chain and thus collapse the mitochondrial intra-membrane potential. This inhibition causes failure to

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provide oxidized ubiquinone to the fourth enzyme in the pyrimidine bio-synthetic pathway, dihydroororate dehydrogenase (PfDHODH) [25, 26], which was validated as an essential enzyme for the parasite and a promis-ing drug target [22, 23, 27]. The search for selective PfDHODH inhibitors that might lead to the new antimalarial therapy remains a current topic amongst antimalarial researchers [22, 28-31].

Aspartate transcarbamoylase (ATC, EC 2.1.3.2) catalyzes the second step of pyrimidine biosynthesis, the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. The ATC from Escherichia coli was fully characterized by William Lip-scomb and colleagues [32]. The E. coli ATC is known to be a highly regu-lated enzyme: the rate of pyrimidine biosynthesis is stimulated or inhibit-ed in response to cellular levels of the end products of the pathway [33]. In E. coli the ATC holoenzyme is composed of six catalytic and six regulatory subunits: three regulatory pairs coordinate two catalytic trimers.

ATCs are also promising targets for anti-proliferative and anti-tumor drugs as Madani and coworkers showed that while almost undetectable in healthy human brain tissue, significant ATC levels were present in all studied tumor samples [34]. In 2014, ATC from P. falciparum (PfATC) was also reported as a potential target for gametocidal drug development [35].

PfATC is a polypeptide of 375 amino acids with a predicted molecular mass of 43.3 kDa (PlasmoDB ID: PF3D7_1344800; Table 1). BLAST analysis of PfATC sequence shows that the N-terminal region of approximately 36 amino acids does not have known structural homologs [36]. In addition no homologs of the ATC regulatory chain have yet been identified in the plasmodial genome. The amino acids (55-372) can be aligned to the cata-lytic domain of P. abyssi ATC [37] with an identity of 38%. Based on the absence of structural analogs in the Protein Data Bank (PDB) and predict-ed presence of apicoplast-targeting signal [38], the N-terminal region of 36 amino acids was removed and a truncated construct PfATC-Met3 was used in this study. Attempts to obtain the full-length PfATC have failed due to spontaneous proteolysis near the N-terminus (data not shown).

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The truncated PfATC (PfATC-Met3) consists of 349 amino acids including 339 of wild type PfATC (37-375) and SAWSHPQFEK sequence of Strep-tag (IBA3) at the C-terminus with approximate molecular mass of 40.3 kDa. Recent attempts to characterize PfATC were published [39, 40], but a crystal structure is necessary to study the molecular basis of the catalytic mechanism, support validation of PfATC as a drug target and potentially support structure based drug design.

In this manuscript, the crystal structure and preliminary characterization of a truncated PfATC as well as its localization within the parasite is re-ported. Structural information was used to design a mutant of PfATC with significantly reduced catalytic activity; the activity profiles of the mutant and wild type enzymes are reported. These data are essential in under-standing the role of plasmodial aspartate metabolism and might lead to a novel antimalarial therapy.

2. Materials and Methods

2.1. Cloning

A full length gene encoding for PfATC was amplified via reverse transcrip-tase PCR using P. falciparum total RNA from P. falciparum as a template. In the first step, the cDNA was synthesized from RNA using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific). In the second step, full-length PfATC gene was amplified from the cDNA tem-plate using sequence-specific sense (5´-GCGCGCGGTCTCCAATGATT-GAAATATTTTGCACTGC-3´) and antisense (5´-GCGCGCGGTCTCCG-CGCTGCTAGTTGATGAAAAAATGAG-3´) primers. The PCR reaction was performed using SuperMix HiFi polymerase mix (Invitrogen). The generated PCR product was digested with BsaI (restriction sites under-lined) and cloned into pASK-IBA3 vector (www.iba-lifesciences.com) previously digested with the same enzyme. The final expression plasmid pASK-IBA3-PfATC-full encoded the full-length version of PfATC with the additional amino acids SAWSHPQFEK (Strep-tag) at the C-terminus (Ta-

ble S1).Cloning of the truncated PfATC (PfATC-Met3) was performed via

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PCR amplification using pASK-IBA3-PfATC-full plasmid as a template, sense (5´-GCGCGCGGTCTCCAATGTTTTATATCAATAGCAAG-3´) and antisense (5´-GCGCGCGGTCTCCGCGCTGCTAGTTGATGAAAAAAT-GAG-3´) sequence-specific primers.

The PCR fragment was similarly cloned into pASK-IBA3 vector previous-ly digested with BsaI. The final expression plasmid pASK-IBA3-PfATC-Met3 encoded the truncated version of PfATC (residues 37-375) with the additional amino acids SAWSHPQFEK (Strep-tag) at the C-terminus (Ta-ble 1). All plasmid samples were verified by sequencing.

2.2. Expression

PfATC-Met3 was recombinantly expressed using E. coli Rosetta 2 (DE3) pLysS (Nalgene) competent cells transformed with pASK-IBA3-PfATC-Met3 expression plasmid. The optimal cell line and inductor concentra-tion were chosen based on preliminary small-scale expression trials (data not shown). The culture was propagated in 1 L of selective LB media sup-plemented with 50 μg ml-1 ampicillin, 35 μg ml-1 chloramphenicol and 4 mM MgCl2 at 310 K in 2 L baffled Erlenmeyer flasks (Nalgene) and in-duced with 60 ng ml-1 of Anhydrotetracycline (AHT) according to the ex-pressiontrial results. The temperature of the culture was lowered to 291 K after induction and cells were harvested by centrifugation after overnight expression. Centrifugation was performed using SLA-3000 rotor (Ther-mo Scientific) at 10,000 x g for 30 min.

2.3. Purification

Purification of recombinant PfATC-Met3 was performed via Strep-Tag chromatography according to the manufacturer’s recommendations (IBA Lifesciences). The bacterial pellet from 1 L culture was resuspended in 40 mL Lysis buffer [50 mM Tris (Tris(hydroxymethyl)aminomethan) pH 8.0, 300 mM NaCl, 5% (v/v) glycerol, 3 mM β-mercaptoethanol (BME)].

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Table 1. Macromolecule production information

Gene Truncated PfATC (wild type)

Source organism Plasmodium falciparum strain 3D7

DNA source pASK-IBA3-PfATC-full plasmid

Forward primer (BsaI) 5´-GCGCGCGGTCTCCAATGTTTTATATCAATAGCAAG-3´

Reverse primer (BsaI)5´-GCGCGCGGTCTCCGCGCTGCTAGTTGATGAAAAAAT-GAG-3´

Expression vector pASK-IBA3

Expression host Escherichia coli

Complete amino acid se-quence of the construct produced

MFYINSKYKIDLDKIMTKMKNKSVINIDDVDDEELLAILYTSKQFE

KILKNNEDSKYLENKVFCSVFLEPSTRTRCSFDAAILKLGSKVLNI

TDMNSTSFYKGETVEDAFKILSTYVDGIIYRDPSKKNVDIAVSSSS

KPIINAGNGTGEHPTQSLLDFYTIHNYFPFILDRNINKKLNIAFVG

DLKNGRTVHSLSKLLSRYNVSFNFVSCKSLNIPKDIVNTITYNLKK

NNFYSDDSIKYFDNLEEGLEDVHIIYMTRIQKERFTDVDEYNQYKN

AFILSNKTLENTRDDTKILHPLPRVNEIKVEVDSNPKSVYFTQAEN

GLYVRMALLYLIFSSTSSAWSHPQFEK

Gene PfATC-R109A/K138A double mutant (PfATC-RK)

DNA source pASK-IBA3-PfATC-Met3 plasmid

Forward primer (R109A)5´-GTTCCTTGAACCAAGTACAGCAACAAGATGTTCTTTTGAT-

GC-3´

Reverse primer (R109A)5´-GCATCAAAAGAACATCTTGTTGCTGTACTTGGTTCAAG-

GAAC-3´

Forward primer (K138A)

5´-CTGATATGAATTCAACTTCTTTTTATGCGGGAGAAACTGTT-

GAAGATGCC-3´

Reverse primer (K138A)5´-GGCATCTTCAACAGTTTCTCCCGCATAAAAAGAAGTTGAAT-

TCATATCAG-3´

Expression vector pASK-IBA3

Expression host Escherichia coli

Complete amino acid se-quence of the construct produced

MFYINSKYKIDLDKIMTKMKNKSVINIDDVDDEELLAILYTSKQFE

KILKNNEDSKYLENKVFCSVFLEPSTATRCSFDAAILKLGSKVLNI

TDMNSTSFYAGETVEDAFKILSTYVDGIIYRDPSKKNVDIAVSSSS

KPIINAGNGTGEHPTQSLLDFYTIHNYFPFILDRNINKKLNIAFVG

DLKNGRTVHSLSKLLSRYNVSFNFVSCKSLNIPKDIVNTITYNLKK

NNFYSDDSIKYFDNLEEGLEDVHIIYMTRIQKERFTDVDEYNQYKN

AFILSNKTLENTRDDTKILHPLPRVNEIKVEVDSNPKSVYFTQAEN

GLYVRMALLYLIFSSTSSAWSHPQFEK

Table 1. The cloning details for truncated version of PfATC-Met3 and the mutant PfATC-RK. BsaI restriction sites in the primers are underlined. Point mutations are shown in bold and highlighted. Additional Strep-tag residues at the C-terminus are shown in bold italic.

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The lysis was performed by sonication on ice. The supernatant contain-ing soluble Strep-tagged PfATC-Met3 was clarified by centrifugation at 45,000 x g (SS-34 rotor, Thermo Scientific) and filtered using 0.45 μm fil-ter membrane (Whatman). The filtered supernatant was incubated with 2.0 mL Strep affinity resin (Strep Tactin Superflow, IBA Lifesciences) for 20 minutes at 277 K and subsequently poured onto a gravity-flow column (Bio Rad) and washed with 100 mL Lysis Buffer. The protein was eluted with 8 mL Elution buffer [50 mM Tris pH 8.0, 300 mM NaCl, 2.5 mM desthiobiotin, 5% (v/v) glycerol, 3 mM BME]. The eluate was pooled and concentrated at 277 K to 2 mg mL-1 using Vivaspin 4 concentration col-umn with a 10 kDa cutoff (Sartorius Stedim Biotech).

In order to select the correct buffer for further purification a Thermal Shift Assay [41, 42] was performed using CFX96 Real-Time system (Bio-Rad). SYPRO Orange dye (5000x stock, Invitrogen) was added to the protein sample (2 mg mL-1) at 1:500 ratio. Each experiment consisted of 5 μL of the protein/dye mixture and 45 μL of the buffer component to be screened. Water was used as a control instead of the buffer sample. Inflection points in graphs of relative fluorescence units (RFU) against temperature were determined manually and used as an indicator of the sample thermal stability in presence of the screened buffer components. Components that exhibited positive thermal shift in comparison to a wa-ter control sample were used to select a size exclusion chromatography (SEC) buffer [20 mM Tris pH 8.0, 300 mM NaCl, 10 mM Na-Malonate, 5% (v/v) glycerol and 2 mM BME]. The remaining protein was concen-trated to a volume of 1 mL (Vivaspin4, Sartorius Stedim Biotech) and purified via SEC using a HiLoad 16/60 Superdex 75 column (GE Health-care) pre-equilibrated with SEC buffer using NGC liquid chromatography system (BioRad). The purified protein eluted as a single peak and was pooled and concentrated to 10 mg mL-1 at 277 K (Vivaspin4, Sartorius Stedim Biotech). The final concentration was determined based on the protein theoretical absorbance at 280 nm [Abs 0.1% (1 mg ml-1) = 0.84;

http://web.expasy.org/protparam].

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Figure 1

Figure 1. (a) 10 % SDS–PAGE of the purified PfATC-Met3. The sample was boiled in SDS-loading buffer prior to loading and the gel was stained with Coomassie Blue. Left lane - unstained protein marker (Thermo Scientific; labelled in kDa), right lane – final PfATC-Met3 sample. (e) A diffrac-tion-quality single PfATC-Met3 crystal mounted in a cryo-loop (Molecular Dimensions Ltd.) grown in 200 mM Sodium Sulphate, 5 mM MgSO4, 15 % (w/v) PEG 3350 in 100 mM bis-Tris Propane pH

7.5. Dimensions as measured on the beamline are shown.

The concentrated protein was immediately used in crystallization trials. The estimated protein purity was better than 95% (Figure 1, a) as assessed by Coomassie Brilliant Blue-stained SDS-PAGE [43]. The final yield of purified PfATC-Met3 was 6 mg per liter of culture. Expression and puri-fication of the mutant version of PfATC-Met3 were performed identically

2.4. Crystallization

Screening for crystallization conditions for PfATC-Met3 was performed using a high-throughput crystallization robot (Gryphon, Art Robbins) against commercially available sparse-matrix screening kits (JCSG plus

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and PACT premier; Molecular Dimensions Ltd.). All experiments were performed at 293 K using the sitting drop vapor diffusion technique in 96-well MRC2 plates (Molecular Dimensions Ltd.). Equal volumes (0.1 μL) of protein solution and crystallization reagent were equilibrated against 50 μL of reservoir solution. Medium-size single crystals appeared overnight in various conditions containing PEG 3350/4000. Further op-timization was performed using the hanging-drop technique by varying the precipitant concentration, ionic strength, pH and buffer conditions. The optimized conditions consisted of equal amounts (1.5 μL) of 9 mg ml-1 PfATC-Met3 sample and 0.2 M Sodium Sulfate, 5 mM MgSO4, 15 % (w/v) PEG 3350 in 0.1 M bis-Tris Propane pH 7.5 as the crystallization solution at 293 K. Rhomboid-shaped diffraction-quality crystals (Figure 1b) with maximum dimensions of 200 μm appeared overnight and were subsequently harvested using mounted round LythoLoops (Molecular Di-mensions Ltd.), incubated in the Cryo-buffer (below) and flash-cooled in liquid nitrogen prior to shipment to the

synchrotron. The Cryo-buffer was chosen based on an estimation from [44] and consisted of 0.2 M Sodium Sulphate, 5 mM MgCL2, 15 % (w/v) PEG 3350, 20% glycerol in 0.1 M bis-Tris Propane pH 7.5.

2.5. Data Collection

Cryo-cooled PfATC-Met3 crystals were sent to European Synchrotron Radiation Facility (ESRF, Grenoble) using dry-shipping cryo-container (Taylor-Wharton) and a 2.4 Å data set was collected at 100 K in the nitro-gen stream at the ID23-2 beamline. Initial characterization of the crystals and the optimization of data collection parameters were performed using BEST software [45, 46]. The space group of PfATC crystals was calculated to be monoclinic P 21 with unit-cell parameters of a = 87.0, b = 103.8, c = 87.1, α = 90.0, β = 117.7, γ = 90.0, the solvent content was calculated to be 59.4 %. The data were processed using the XDSapp [47] software. The analysis also showed that the crystal was twinned. The Matthews co-efficient [48] predicted a trimeric form of PfATC-Met3 in the asymmetric

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unit. The data collection and processing statistics are reported in Table 2.

2.6. Data processing and Refinement

The structure of PfATC-Met3 was solved by molecular replacement using BALBES [49] software within the CCP4 package [50]. The crystal structure of ATC from Pyrococcus abyssi [37] was used as a search model (38% identity) yielding a solution of three molecules in the asymmetric unit. The model was further optimized via manual rebuilding in COOT [51] and refined with REFMAC5 software [52]. The final refinement steps were carried out with global NCS restraints, TLS restraints calculated via TLSMD web server [53, 54]. These steps also included twin-refinement.

The final 2.5-Å model of PfATC-Met3 consisted of 3 molecules in the asymmetric unit with R factor of 0.18 and free R factor of 0.22 (Table 3) and has been deposited in the Protein Data Bank (PDB) with the accession code 5ILQ. The structure figures in this manuscript were prepared with PyMol [55].

2.7. Mutagenesis

The double mutant PfATC-Met3-R109A/K138A (hereafter PfATC-RK) was created via site-directed mutagenesis using the previously described pASK-IBA3-PfATC-Met3 expression plasmid as a template. The PCR re-action was performed using Phusion Hot Start II DNA polymerase (Ther-mo Scientific) according to the manufacturer’s recommendations. For mutagenesis details please refer to Table 1. The generated mutant plas-mid was treated with DpnI enzyme for 2 hours at 310 K to eliminate the parental template. The mutations were validated by sequencing. Similarly to the wild type the mutant construct also encoded a C-terminal Strep-tag. Expression and purification of PfATC-RK mutant were performed identi-cally to PfATC-Met3.

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Table 2. Data collection and processing

Values for the outer shell are given in parentheses.

Diffraction source ESRF ID 23-2

Wavelength (Å) 0.98

Temperature (K) 100

Detector PILATUS 6M

Crystal-detector distance (mm) 337.55

Rotation range per image (°) 0.1

Total rotation range (°) 129

Exposure time per image (s) 0.04

Space group P 21

a, b, c (Å) 86.96, 103.80, 87.11

α, β, γ (°) 90.0, 117.6, 90.0

Mosaicity (°) 0.2

Resolution range (Å) 45.0 – 2.42 (2.56 – 2.42)

Total No. of reflections 126651 (20324)

No. of unique reflections 50423 (8051)

Completeness (%) 95.9 (95.6)

Redundancy 2.51 (2.52)

<I/σ(I)> 10.81 (1.98)

CC1/2 99.7 (76.6)

Rmeas(%) 7.6 (58.0)

Overall B factor from Wilson plot (Å2)

56.64

Rmeas is defined as Σhkl Σi | Ii(hkl) - <I(hkl)> | / Σhkl Σi Ii(hkl), where Ii(hkl) is the ith intensity measurement of reflection hkl and <I(hkl)> is the average intensity from multiple observations.

The free R-factor was calculated using 5% of randomly selected reflections omitted from the refinement.

2.8. Activity Assays

The kinetic properties of PfATC-Met3 as well as PfATC-RK were investi-gated according to [56], [57] and [58] with minor modifications. Briefly, the reaction was carried out at room temperature in a total volume of 160

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μl of 14 mM L-aspartate and 1 mM carbamoyl phosphate (CP) saturated substrate solution in 200 mM Tris-Acetate buffer pH 8. The reaction was stopped by 80 μl of 25 mM Ammonium molybdate in 4.5 M H2SO4. After all the reactions were stopped 160 μl of 0.5 μM malachite green in 0.1 % (w/v) poly(vinyl alcohol) (PVA) were added and incubated for 30 min at room temperature. Subsequently, the absorption of the samples at a wavelength of 620 nm was measured. The analyses were evaluated from at least three independent quadruplicate assays using GraphPad Prism 4 (GraphPad Software, USA).

2.9 Transfection

The open reading frame of the full-length PfATC was cloned in front of the GFP gene of the expression vector pARL1a and subsequently trans-fected into P. falciparum. Briefly, erythrocytes were washed in cytomix [59] and an aliquot of 450 ml was combined with approximately 100 mg of the construct resuspended in 50 ml of TE buffer. The red blood cells were electroporated using a Gene-Pulser X-Cell total system (BIORAD) at 0.31 kV and 900 mF. After electroporation the cells were transferred into pre-warmed RPMI medium and inoculated with 50 ml of red blood cells infected with 10% schizonts to give a parasitaemia of 1%. Four hours post transfection the culture medium was exchanged. Parasites were grown for 24 h without drug selection before the medium was supplemented with 5 nM of WR 99210. Parasites were first observed after 16–60 days of se-lection and live parasites were analyzed by fluorescent microscopy using an Axioskop 2 plus microscope (ZEISS). In order to visualize the nucleus, parasites were incubated with Hoechst 33342 dye according to the manu-facturer’s recommendation (Invitrogen). For Co-localization experiments to visualize the ER, transfected Plasmodium was supplemented with 2 mM of ER-Tracker™ Red BODIPY-TR (Invitrogen) prior to microscopy.

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3. Results

A truncated version of ATC from Plasmodium falciparum was cloned, recombinantly expressed, purified and crystallized. Full-length PfATC is a protein of 375 residues and calculated molecular weight of 43.3 kDa. BLAST [36] analysis has revealed no structural homologs of the first 36 N-terminal residues (Figure 2). PfATC shows 38% identity (84% query cover) with the catalytic subunit of ATC from Pyrococcus abyssi (PDB code: 1ML4; [37]) and 39% identity (84% query cover) with the Esche-richia coli ATC (PDB code: 1D09; [60]). Nucleotide BLAST analysis also shows 37% identity (86% query cover) with ATC domain of Human CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) protein. The crystal structure of this domain is not yet available, although the preliminary data have been reported [62]. Fur-ther analysis with PlasmoAP tool [38] showed a strong prediction for the N-terminal region to contain an apicoplast targeting sequence with a possible cleavage site between residues 27-28 (IRT-KK) (Table S1). The GFP-labelling experiments in vivo showed distinct localisation of PfATC rather than in ER supporting the hypothesis of apicomplexian nature of the enzyme (Figure 3). Based on these data, the third methionine in the sequence (Met37) was selected as a starting point of our construct.

Attempts to express and purify full-length PfATC with sufficient purity have failed. SDS-PAGE analysis of the elution fraction of Strep-purifi-cation showed three distinct Strep-tagged (C-terminal) bands with sizes around 43 kDa and further size-exclusion purification showed three over-lapping peaks that could not be separated with sufficient purity (data not shown). Use of protease-inhibitors cocktail during the lysis and purifica-tion did not show any significant improvement. The presence of at least three versions of PfATC of different lengths in the elution fraction suggests that the full-length protein is proteolyzed near the N-terminus due to the apicoplast-targeting nature of this region. The activity assay performed with PfATC-Met3 showed that it possesses catalytic activity and further experiments have been performed with this truncated PfATC construct.

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Figure 2

Figure 2. The homology analysis of PfATC-Met3 was performed using BLAST tool [36] and visu-alized via Tcoffee [61]. Red, yellow, green and blue colours represent good, average, bad and weak alignment, respectively. Residues essential for the active sites are shown with arrows. Absolutely, strongly and slightly conserved residues are marked with ``*``, ``:`` and ``.``symbols, respectively.

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Table 3. Structure solution and refinement

Resolution range (Å) 20.0 – 2.5

Completeness (%) 95.2

σ cutoff 1

No. of reflections, working set 42863

No. of reflections, test set 2354

Final Rcryst 0.18

Final Rfree 0.22

No. of non-H atoms 7909

Protein 7801

Ion 23

Water 88

R.m.s. deviations

Bonds (Å) 0.019

Angles (°) 1.96

Average B factors (Å2) 57.39

Protein 58.04

Ion 56.76

Water 51.47

Ramachandran plot

Most favoured (%) 93.60

Allowed (%) 5.98Free R-factor was calculated using 5% of randomly selected reflections omitted from the refinement.

Values in parentheses correspond to the highest resolution shell.

The recombinant PfATC-Met3 was produced with a C-terminal Strep-tag (Iba Lifesciences) and purified by Strep-chromatography according to the manufacturer’s recommendations. In order to achieve higher purity and homogeneity, PfATC-Met3 was further purified via SEC and the final pu-rity was assessed via SDS-PAGE analysis (Figure 1, a). The yield of pure homogeneous protein was approximately 6 mg per liter of culture. In or-der to increase the post-purification stability of the protein sample and increase crystallization chances, the SEC buffer was chosen based on the results of Differential Scanning Fluorimetry. The fresh purified protein was concentrated to 9 mg mL-1 and used for crystallization experiments.

Multiple crystals of PfATC-Met3 appeared overnight in various crys-

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tallization screening conditions containing PEG (polyethyleneglycol) 3350/4000. Optimization of the initial conditions yielded single PfATC crystals with maximum dimension of 0.2 mm (Figure 1, b). The final crys-tallization condition was 0.1 M bis-Tris Propane pH 7.5, 0.2 M Na-Sulf-fate, 5 mM Mg-Sulfate and 15% (w/v) PEG 3350.

The optimized crystals diffracted X-rays to a maximum resolution of 2.4 Å at ID23-2 beamline (ESRF, Grenoble). The space group of the PfA-TC-Met3 crystals was determined to be monoclinic P 21, with unit-cell pa-rameters of a= 87.0, b= 103.8, c = 87.1, α = 90.0, β = 117.7, γ = 90.0. The presence of three PfATC molecules per asymmetric unit was confirmed by molecular replacement and the 2.5-Å structure of PfATC-Met3 has been deposited in the PDB with the accession code 5ILQ.

3.1. Overall structure of PfATC-Met3

PfATC-Met3 is a homo-trimer with three active sites formed at the oligo-meric interfaces (Figure 4, a, b). Superposition of the PfATC-Met3 struc-ture and catalytic subunit of E. coli ATC (PDB code: 1D09; [60]) showed high level of sequence and secondary structure conservation (Figure 2, Figure 5). BLAST [36] analysis against nine known homologs (structures available) showed that of 375 residues 46 (12.3%) were absolutely con-served, 43 (11.5%) were strongly conserved and 25 (6.7%) slightly con-served. The loops formed by 84-91, 203-212 and 265-275 residues of PfA-TC-Met3 are slightly longer than their E. coli analogues (residues 34-36, 150-155 and 206-208, respectively). In addition, the N-terminal region of PfATC-Met3 model (residues 37-48) is also longer and shows no second-ary structure. Poor electron density coverage suggests that this region is highly mobile. For this reason N-terminal residues 37-46 (chain A) and 37-43 (chains B, C) were excluded from the final model as well as the loops formed by the residues 297-311 (all three chains) and C-terminal region of chain A (residues 375-378). The presence of additional electron density near the C-terminus was modelled as the Strep-tag (SAWSHPQFEK) with

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the last 2-3 residues (FEK) excluded.

In 1991 Stevens and co-workers reported the essential residues that form the active site of ATC based on the structure of E. coli ATC, confirmed by site-specific mutagenesis experiments [65]. In PfATC-Met3, all the ho-mologous residues are absolutely conserved (BLAST) among the ATC´s from other organisms (Figure 2, 6). Each subunit of the trimer hosts an active site comprised with two residues (Ser135 and Lys138) from the ad-jacent chain.

PISA analysis [66] of the PfATC-Met3 structure showed that the oligo-meric contact between each subunit pair (hereafter A and B) is formed by 32 (Buried Surface Area (BSA) of 899 Å2) and 27 residues (BSA of 956 Å2), respectively (Figure 7, a-f). The interface from the subunit A consists of 9 absolutely conserved, 3 strongly conserved and 2 slightly conserved resi-dues. The adjacent interface B has 8 absolutely, 4 strongly and 2 slightly conserved residues, respectively. Each subunit has a total surface area of approximately 14,300 Å2 where 1855 Å2 (13%) belong to the intraoligo-meric interfaces.

Figure 3. Fluorescent microscopy imaging of P. falciparum transfected with GFP-labeled PfATC shows distinct localisation of PfATC (see materials and methods 2.9).

Figure 4. The overall model of PfATC-Met3. (a) PfATC-Met3 is a homo-trimer with three active sites (shown with stars) formed at the oligomeric interfaces. (b) Schematic view of PfATC homo-trimer.

Figure 5. The secondary structure of PfATC-Met3 compared to the E. coli ATC (a) and B. subtlis ATC (b). Coordinates of PfATC-Met3 (green) structure and catalytic subunits of E. coli ATC in liganded R-state (magenta, PDB code: 1D09, [60] and unliganded T-state B. subtlis ATC (blue, PDB code:

3R7D, [63]) were superimposed using COOT [51, 64].

183

Figure 3

Figure 4

Figure 5

184

Figure 6

Figure 6. Superposition of the key active site residues of PfATC-Met3 (green) and E. coli ATC (ma-genta, PDB code: 1D09) that exhibit the absolute conservation amongst homologous species (see Fig-

ure 2).

Figure 7

Figure 7. PfATC-Met3 structure analysis reveals that each subunit forms two oligomeric contacts with other subunits of the trimer (a). Residues that form these contacts are shown in blue (b-f). Ab-solutely (red), strongly (orange) and slightly conserved (green) residues of these interfaces are shown (c,e). Active site residues (magenta) are also shown on the surface (f).

185

3.2. Mutagenesis studies and activity profile of the truncated version of PfATC

Based on the mutagenic studies on E. coli ATC summarized in [32] and overall structure similarity, a mutant PfATC-Met3 version was construct-ed. In E. coli, point mutations of the essential active site residues of the catalytic chain ATC resulted in significant loss of activity. For example, guanidinium group of Arg54 of E. coli ATC was shown to be crucial for binding of the substrate carbamoyl-phosphate (CP) and enzymatic con-densation of CP and aspartate, and when removed (Arg54Ala mutation) caused 17,000-fold loss in activity and 13-fold reduction in affinity for CP [67, 68]. The amino group of Lys84, that completes the active site of the adjacent subunit, interacts with both Arg54 and aspartate upon binding; Lys84Asn mutation has resulted in 1200-fold activity loss of the E. coli enzyme [60]. Both mutations did not affect the folding and the structure of the enzyme as confirmed by X-ray data (data not shown). Comparison between the crystal structures of PfATC-Met3 and the E. coli ATC (PDB code: 1D09; [60]) showed that the Arg109 and Lys138 residues of the ac-tive site of PfATC-Met3 are homologous to Arg54 and Lys84 of E. coli ATC. The mutant version PfATC-RK (R109A/K138A) was designed and cloned using site-specific mutagenesis technique and recombinantly ex-pressed according to the same protocol as the PfATC-Met3 (See Materials and Methods section).

In order to confirm the presence of the catalytic activity of the recom-binant PfATC-Met3 a phosphate-detection system based on malachite green [57] was established. The specific in vitro activity of PfATC-Met3 was 11.04 ± 1.04 μmol min−1 mg−1. Activity assays with the double mutant PfATC-RK was also performed and showed significantly lower value of 0.85 ± 0.30 μmol min−1 mg−1. Additional native PAGE electrophoresis and DLS (Dynamic light scattering) experiments (data not shown) confirmed the presence of oligomeric PfATC-Met3 assembly in the solution for both wild type and mutant versions. These data have confirmed the initial hy-pothesis that the mutations of the key active site residues (R109A and

186

K138A) significantly reduce the catalytic activity while having no adverse effect on the oligomeric assembly.

4. Discussion

Pyrimidine biosynthesis of P. falciparum represents highly attractive tar-

get for future antimalarial drug development. Determined crystal struc-

ture of truncated aspartate transcarbamoylase from Plasmodium falci-

parum, the enzyme that catalyses the second step of the pathway, shows

high degree of evolutional conservation amongst its homologs from other

organisms. The active site of the enzyme shows the highest degree of con-

servation, making the structure-based design of specific active site target-

ing inhibitors of PfATC and its further validation as a drug target highly

challenging.

187

Table S1. Macromolecule production information (Full-length PfATC)

Source organism Plasmodium falciparum strain 3D7

DNA source cDNA generated from total RNA via reverse transcriptase PCR

Forward primer (BsaI) 5´-GCGCGCGGTCTCCAATGATTGAAATATTTTGCACTGC-3´

Reverse primer (BsaI) 5´-GCGCGCGGTCTCCGCGCTGCTAGTTGATGAAAAAATGAG-3´

Expression vector pASK-IBA3

Expression host Escherichia coli

Complete amino acid sequence of the con-struct produced

MIEIFCTAIVVITILIVGVFVYMIIRTKKKKLKLDNMFYINSKYKI

DLDKIMTKMKNKSVINIDDVDDEELLAILYTSKQFEKILKNNEDSK

YLENKVFCSVFLEPSTRTRCSFDAAILKLGSKVLNITDMNSTSFYK

GETVEDAFKILSTYVDGIIYRDPSKKNVDIAVSSSSKPIINAGNGT

GEHPTQSLLDFYTIHNYFPFILDRNINKKLNIAFVGDLKNGRTVHS

LSKLLSRYNVSFNFVSCKSLNIPKDIVNTITYNLKKNNFYSDDSIK

YFDNLEEGLEDVHIIYMTRIQKERFTDVDEYNQYKNAFILSNKTLE

NTRDDTKILHPLPRVNEIKVEVDSNPKSVYFTQAENGLYVRMALLY

LIFSSTSSAWSHPQFEK

Table S1. The cloning details for the full version of PfATC. BsaI restriction sites in both primers are underlined. Predicted apicoplast targeting sequence (PlasmoAP, [69]) is also underlined. Predicted cleavage site (IRT-KK) is shown in italic. Additional Strep-tag residues at the C-termi-nus are shown in bold italic.

188

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